Eclipse+ NMR Training Guide

Eclipse+ NMR Training Guide Version 4.3.4

ECLIPSE+ NMR Training Guide Revision 20050617 Copyright 2005 by JEOL USA, Inc. Analytical Instruments Division 11 Dearborn Road Peabody, MA 01960 (978) 535-5900 All Rights Reserved This document has been carefully checked and is believed to be correct and reliable. However, JEOL USA and their subsidiaries assume no responsibility for errors. Statements in this document are not intended to create any warranty or specification, expressed or implied. Specifications and performance characteristics of the hardware and/or software described in this manual may be changed at any time without notice. JEOL USA and their subsidiary companies reserve the right to make changes in any products listed. JEOL USA and their subsidiaries do not assume any liability arising out of the application or use of any statement, recommendation, product, or circuit described herein. This document does not convey a license of any patent rights pertaining to JEOL USA, or their subsidiaries; neither does this document convey a license of any patent rights of others. Discussion of any particular hardware or software feature or option in this document does not imply the feature is available and/or standard on any instrument. All specifications are subject to change ECLIPSE+ NMR Training Guide, v4.3.4 ii

Table of Contents I. Shimming the System to Specifications 5 Lineshape Test 7 II. Saving Pulse Widths 10 1 H Obs 90 11 13 C Obs 90 15 1 H Decoupling 90 18 DEPT 90 Check 20 13 C Inverse 90 and Decoupling 90 21 1 H Selective 90 23 1 H Spin-lock 90 25 19 F Obs 90 27 31 P Obs 90 29 15 N Obs 90 31 III. Commonly Used Experiments in Structure Elucidations 33 1D Proton 34 Loading a Process List 34 Using the Pointer Tools to Manipulate Data 35 Using the Peak Mode of the Pointer Tool 36 Using the Integral Mode of the Pointer Tool 38 Plotting Data 39 1D Carbon and DEPT 135 41 DQF-COSY 44 Loading High-Resolution Projections in Homonuclear Data 44 HMQC 48 Loading High-Resolution Projections in Heteronuclear Data 48 HETCOR 50 Edited HSQC 53 Phasing 2D Data 53 1 H- 15 N Correlations with HMQC 56 HMBC 58 1D DPFGSE-NOE 61 ROESY 63 IV. Using Preferences to Customize an Account 66 V. File Management in Delta 67 Setting default paths with the Preferences Tool 67 Using File Manager 67 Deleting Processed Data with File Manager 68 Performing a File Search with File Manager 69 Comparing data using File Information 70 ECA/ECX NMR Training Guide, v4.3-a 3

VI. Shutting Down/Starting up Your System 71 Shut Down 71 Start-up 71 Restarting Control Only 72 VII. NMR System Backup/Restore with Delta Spectrometer Backup 73 Windows/Mac 73 LINUX 73 VIII. Filling Cryogens 74 Cryogen Sensors 74 Nitrogen Fills 74 Helium Fills 75 Appendix A Shimming the Non-Spin Shims 76 Appendix B Preparing the System for Gradient Shimming 77 Deuterium Grad Shim Pulse Width 90 79 Generating 2H Gradient Shimming Basis Sets 79 Configuring Delta for Gradient Shimming Integration 80 Appendix C Helium Transfer Dewar Levels 81 ECLIPSE+ NMR Training Guide, v4.3.4 4

I. Shimming the System to Specifications Purpose: Obtain the system shims and store the maps Sample needed: 1% CHCl 3 in acetone d-6 lineshape sample Instrument preparation: A deuterium gradient shim pulse width and calibration maps must be present. Note that the pulse width and maps are measured by the JEOL service engineer at installation. These maps and pulse width usually need not be re-measured. Open Spectrometer Control -> Config -> Gradient Shim Tool. Click. Set Iterations to 2. Select Z1-Z6 of the Shim Set. Click and. Click. The data acquisition should start. If you need to stop the experiments from running, click Abort! (Figure 1). One can monitor the Figure 1 Click Abort! to terminate gradient shimming events ECA/ECX NMR Training Guide, v4.3-a Figure 2 The gradient image obtained during gradient shimming progress of the experiments by looking at the View Tool. In the Processing menu of the View Tool click Abs. The resulting image should eventually look something like Figure 2. To help judge whether the shims are converging, look at the residual window. The 5

residual window displays both the changes in the shims and the changes in the residual. Figure 3 shows a typical result. When the shimming process completes, relock the sample. Turn off the spinner and shim the non-spin shims. You can shim in the order given in the Shim Groups portion of the Sample Tool (Figure 4), or use the abbreviated procedure given in Appendix A. If there are significant changes made to the non-spin shims, rerun gradient shimming. Otherwise, turn the spinner back on and optimize the Lock Phase (Figure 5) by adjusting to get the maximum lock signal. To verify that the shims are optimized you need to run the Lineshape Test. Figure 3 The gradient shimming residual window (3 iterations) Figure 4 Click on the double-arrow to change the shims visible in the Sample Tool Figure 5 Adjust lock Phase in the Lock Control section of the Sample Tool ECLIPSE+ NMR Training Guide, v4.3.4 6

Lineshape Test Purpose: Determine if the shims are within specifications, if acceptable save the shims Sample needed: 1% CHCl 3 in acetone d-6, lineshape sample Instrument preparation: A very well shimmed system. Experiment used for test: /usr/delta/global/experiments/service/1h_lineshape.exp Process list used for test: dc_balance, fft Set recvr_gain to a value that does not result in clipping. The T 1 s of the chloroform proton are on the order of 50[s], so auto_gain can give poor results. Moreover, it is important that the spins be fully relaxed to obtain an accurate result in this test. Run the experiment on your well-shimmed sample. Using the Zoom data function of the pointer tool, zoom and inspect the main peak. This peak should be symmetric and should not be split near the top. If it is split at the top, you may need to touch up Z1. Next, the width of the peak needs to be measured. Because the shape of an NMR signal can vary widely with shimming, the peak widths in this test are measured relative to the 13C satellites. Zoom the peak so the 13 C satellites are visible. In acetone, they should be about 210[Hz] apart. With the cursors function of the pointer tool, use Create cursor to place a cursor at the height of the 13 C satellites, and then one at one-fifth that height (Figure 6). One can estimate these values given the intensity (!I) displayed by the cursor. If the satellites happen to be at different intensities, choose the one of lower intensity. Now, zoom the main peak (you can get Zoom data back temporarily by holding down the Shift key) and using Create Measure, measure the width of the main peak where it intersects the height of the 13 C satellites. Take note of this value. Now measure the width at one-fifth the height of the 13 C satellites. If the lineshape specification is met, you should save the shims as the system shims. Select Spectrometer Control -> Tools -> Mode -> Console. Type console into the box that appears. You should notice that the connection in Spectrometer Control changes from Connect to Console. In the Sample Tool make sure, which is the System Shims toggle, is depressed. Click on, which is the save shims to disk icon. Respond to the confirmation window. A message verifying that you saved the system shims will be reported in the Master Console. ECA/ECX NMR Training Guide, v4.3-a 7

Figure 6 Placing cursors at the height of the 13 C satellites, and at one-fifth that height ECLIPSE+ NMR Training Guide, v4.3.4 8

Figure 7 Determining the peak width using Measure. ECA/ECX NMR Training Guide, v4.3-a 9

II. Saving Pulse Widths with Machine Config? You can access this GUI tool in Spectrometer Control by first logging in as console. You need to choose Tools-> Mode-> Console and to type the console password into the box that appears. Press Enter. After a few moments, you will notice the mode will change from "Connect:" to "Console:" and the field containing this text will change color. Now, if you choose the Tools menu, you will have more choices, including Machine Config?. Choose this option. The box in Figure 8 will appear. Choose the probe file you wish to Figure 8 Graphical method for editing files on the acquisition computer Figure 9 Example of a probe file in Machine Config? edit. Note that the probe number for the currently installed probe is indicated in the Sample Tool as the Probe ID. Click Edit. Another box will appear (Figure 9). With this tool, you can edit a particular pulse width by clicking on it, highlighting its numerical value and then re-typing it. If you want to save the change(s) you made, click on Save and then Close. If you make a mistake, you can click on Undo. Each of the subsequent pulse calibration sections indicates where to change the values in this tool. To save pulse widths via command line, open a UNIX shell. Perform an rlogin to the acquisition computer and edit the appropriate file in: /eclipse/probes/. The three entries listed correspond to the full-power obs 90, high-power decoupling 90 and low-power decoupling 90, respectively, for a given domain. ECLIPSE+ NMR Training Guide, v4.3.4 10

1 H Obs 90 Purpose: Determine the proton observe 90 pulse width Sample needed: 5% CHCl 3 with Cr(acac) 3 in acetone d-6 Instrument preparation: Shim and tune probe to the sample ( 1 H). Experiment used: /usr/delta/global/experiments/service/obs90_check.exp /usr/delta/global/experiments/service/obs90_array.exp Process list used: /usr/delta/global/process_lists/std_proton.list A survey spectrum needs to be collected first to get the exact position of the CHCl 3 peak and a receiver gain value. Using obs90_check.exp, run an experiment with auto_gain checked. Also, click force_tune to allow probe tuning. Click on Submit to initiate the experiment and note the gain value reported in the Master Console. You will enter this receiver gain value into the box for recvr_gain in obs90_array.exp. When the data from obs90_check.exp returns, process the data and zoom the chloroform peak. With the Copy position of nearest peak tool, click on the center of the chloroform peak (Figure 10). This will store the peak position in a cut/paste buffer. Move the mouse into the x_offset field in obs90_array.exp, double-click the right mouse button to clear the present offset value and then click the middle mouse button to paste the new offset value. Enter the receiver gain noted previously from the Master Console into the recvr_gain field. Arrayed experiments will not run with auto_gain checked. Submit the arrayed experiment. When the acquisition finishes, the data will be returned in an nd Processor. Click on the ECA/ECX NMR Training Guide, v4.3-a button. A 1D processor will open with the first slice of the data set. Process and manually phase the data set using the buttons for p0 and p1 or click on, which is the autophase button. When phasing is complete, close the 1D processor. You will notice an updated processing list in the nd processor, which will include your phase correction values. These values will be applied to each slice of the data. Now click, which will process the data and display it in a Data Slate (Figure 11). The data in the data slate will be displayed in image mode. Image mode displays the data as if you were looking down on top of the data, stacked side-by-side. The y-axis in this case is time (µs) and the x-axis is in ppm. Zoom the stripe centered around 8 ppm. Now using Pick position (not Copy position of nearest peak ), click on the image view representation at the exact position you put the x_offset. A curve will be generated from the maxima and minima of the peak intensities. Now click on Expansion -> Linearize. This will display the data from head to tail. Determine the best 360º null either from the sinusoidal curve or from the head-to-tail view. After determining what the value in µs is for the null, divide by four to get the 1 H obs 90 pulse width. If you did not reach a 360, modify the array settings in the x_90_width field and rerun the experiment. For display purposes, one can remove all but the curve and head-to-tail views. Move the mouse on top of a view to be removed. Hit the PageUp key on the keyboard to select the view. Notice that the mouse pointer will be colored yellow when passed over the active pane. Click on to remove the view. Figure 12 shows a typical 1 H Obs 90 pulse width result. 11

Figure 13 highlights where to save your pulse width and attenuator values in Tools -> Machine Config?. Figure 10 Finding the x offset for the 1 H Obs 90 determination 1. ECLIPSE+ NMR Training Guide, v4.3.4 12

Figure 11 Image mode representation of data Figure 12 1 H Obs 90 pulse width ECA/ECX NMR Training Guide, v4.3-a 13

* Figure 13 Change the highlighted value to save the 1 H Obs 90 pulse width ECLIPSE+ NMR Training Guide, v4.3.4 14

13 C Obs 90 Purpose: Determine the carbon observe 90 pulse width Sample needed: 50% CHCl 3 with Cr(acac) 3 in acetone d-6 Instrument preparation: Shim and tune probe to the sample ( 13 C) and ( 1 H). Experiment used: /usr/delta/global/experiments/single_pulse_dec.exp Process list used: /usr/delta/global/process_lists/std_carbon.list A survey spectrum needs to be collected first to get the exact position of the CHCl 3 peak and a receiver gain value. In single_pulse_dec.exp, run an experiment with auto_gain checked. Also change scans to 1, x_prescans to 0 and x_angle to 90[deg]. Click on Submit to initiate the experiment and note the gain value reported in the Delta Console. When the experiment returns and finishes processing, zoom the chloroform peak. With the Copy position of nearest peak tool, choose the main of the peak at ~78[ppm] (Figure 14). Paste this value into the x_offset field in single_pulse_dec.exp. Also enter 2.Figure 14 Finding the x offset for the 13 C Obs 90 determination Figure 14 Finding the x offset for the 13C Obs 90 determination the receiver gain value previously noted into the box for recvr_gain. Uncheck auto_gain. Click on to open an array box. Click Y to indicate a pseudo second 15 ECA/ECX NMR Training Guide, v4.3-a

dimension, uncheck Listed, and then enter start, stop and step values of 2, 50 and 2 [us], respectively (Figure 15). Click Set Value. Set relaxation_delay to 20[s]. Submit the experiment. Process the arrayed data, as in the 1 H Obs 90 determination and determine the best 360º null. If you did not reach a 360, modify the array settings in the x_90_width field and rerun the experiment. Figure 16 shows a typical result. Figure 17 highlights where to save your pulse width and attenuator values in Machine Config?. Figure 15 Arraying x_90_width ECLIPSE+ NMR Training Guide, v4.3.4 16

Figure 16 13 C Obs 90 pulse width * Figure 17 Change the highlighted value to save the 13 C Obs 90 pulse width ECA/ECX NMR Training Guide, v4.3-a 17

1 H Decoupling 90 Purpose: Determine the proton decoupling 90 pulse width Sample needed: 50% CHCl 3 with Cr(acac) 3 in acetone d-6 Instrument preparation: Shim and tune probe to the sample ( 13 C, 1 H) Experiment used: /usr/delta/global/experiments/service/irr90_check.exp Process list used: /usr/delta/global/process_lists/std_carbon.list Copy the x_offset value used in the 13 C obs 90 pulse width determination into x_offset and the x_offset value used in the 1 H obs 90 pulse width determination into irr_offset. Enter a value of 1[us] for irr_pulse but remember the current pulse width. Click auto_gain. Submit the experiment. Enter the current pulse width into irr_pulse, and the receiver gain determined from the first experiment into recvr_gain. Uncheck auto_gain. Submit the experiment. Process the 1[us] data so that the peaks at ~78[ppm] are antiphase. Phase the data either manually or with autophase and apply the phase conditions to the process list. Hit Process. Using drag-and-drop, transfer this processing list to the data from the second experiment. The pulse width is a 90 when the two peaks are nulled. If the two peaks still have intensity with the same phase as the 1[us] data, the pulse width is too short. If the peaks flip over, the pulse width is too long. Figure 18 shows a typical result. If you are happy with the result save the pulse width in Machine Config?. Otherwise, run single experiments until you find a good pulse width value. Figure 19 highlights where to save your pulse width value in Machine Config?. Figure 18 1 H Decoupling 90 Pulse width, (top) 1[us] data, (bottom) irr_pulse with a well calibrated 90 ECLIPSE+ NMR Training Guide, v4.3.4 18

* Figure 19 Change the highlighted value to save the 1 H Decoupling 90 pulse width ECA/ECX NMR Training Guide, v4.3-a 19

DEPT 90 Check Purpose: Verify that the proton decoupling 90 pulse width is correct Sample needed: 10% ethylbenzene in chloroform-d Instrument preparation: Shim and tune probe to the sample ( 13 C, 1 H) Experiment used: /usr/delta/global/experiments/service/dept_cal.exp Process list used: /usr/delta/global/process_lists/std_carbon.list Change selection_angle to 90. Set recvr_gain to an appropriate value. Submit the experiment. If the 1 H decoupling 90 pulse width is correct, one should only see the three aromatic carbon peaks. Figure 20 shows a result of a properly calibrated pulse width. Figure 20 DEPT 90 Check for a Well-Calibrated 1 H Decoupling 90 ECLIPSE+ NMR Training Guide, v4.3.4 20

13 C Inverse 90 and Decoupling 90 Purpose: Determine the carbon inverse 90 and decoupling 90 pulse width Sample needed: 5% CHCl 3 with Cr(acac) 3 in acetone d-6 Instrument preparation: Shim and tune probe to the sample ( 13 C, 1 H) Experiment used: /usr/delta/global/experiments/1d_inverse/hmqc_irr_check.exp /usr/delta/global/experiments/1d_inverse/hmqc_irr_dec_check.exp Process list used: /usr/delta/global/process_lists/std_proton.list Copy the x_offset value used in the 1 H obs 90 pulse width determination into x_offset and the x_offset value used in the 13 C obs 90 pulse width determination into irr_offset. Enter a value of 1[us] for irr_pulse but remember the current pulse width. Click auto_gain. Submit the experiment. Enter the current pulse width into irr_pulse, and the receiver gain from the first experiment into recvr_gain. Uncheck auto_gain. Submit the experiment. Process the 1[us] data so that the main peak is ~90 out of phase. This will result in the satellites, which are the peaks of interest, being anti-phase. Apply the phase conditions to the process list. Click Process. Using drag-and-drop, transfer this processing list to the data from the second experiment. Click Process. Zoom the two data sets identically. Note that it is possible to drag x and y limits when the Zoom data cursor is selected. The pulse width is a 90 when the two small peaks are nulled. If the peaks still have intensity with the same phase as the 1[us] data, the pulse width is too short. If the peaks flip over, the pulse width is too long. Figure 21 shows a typical result. If you are happy with the result save the pulse width in Machine Config?. Otherwise, run single experiments until you find a good pulse width value. Figure 22 highlights where to save your 13 C Inverse 90 Pulse width in Machine Config?. To find the decoupling pulse width, use the same procedure except use hmqc_irr_dec_check.exp. Figure 23 shows where to save your 13 C Decoupling 90 Pulse width in Machine Config?. ECA/ECX NMR Training Guide, v4.3-a 21

Figure 21 13 C Inverse 90 Pulse width, (left) 1[us] data, (right) a well-calibrated 90 * Figure 22 Change the highlighted value to save the 13 C Inverse 90 pulse width * Figure 23 Change the highlighted value to save the 13 C Decoupling 90 pulse width ECLIPSE+ NMR Training Guide, v4.3.4 22

Selective 90 Purpose: Calibrate the selective 90 degree pulse width. Sample needed: 5% CHCl 3 in acetone d-6 doped with trace Cr(acac) 3 Instrument preparation: A well-shimmed system with the probe tuned to the sample ( 1 H). Experiments used in this calibration: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/service/20db_att/obs90_w_20db_att.exp This pulse width is necessary for the dpfgse_noe_pfg_s.exp. A survey spectrum on proton needs to be run first. Run the survey spectrum using single_pulse.exp. Transfer the receiver gain value to recvr_gain and the on resonance condition for the CHCl 3 peak to x_offset, in obs90_w_20db_att.exp. Set obs_attenuator to 40[dB]. Set auto_dwell to false as in the spin-lock 90 calibration. Array x_pulse from 2-50[ms] with a step size of 2[ms]. When the data set returns, process it as in the 1 H obs 90 pulse width calibration. You are looking for the best null. Divide the null by two to get your pulse width. Save the pulse width and attenuator value in the dpfgse_noe_pfg_s.exp itself. Note: The optimum pulse width is highly dependent on the amount of bandwidth you require. Recall that the B 1 field can be calculated by: B 1 (Hz) = 1/(4*pulse width [s]). One can conveniently double the effective bandwidth by halving the selective 90 pulse width and lowering the obs_attenuator value by 6[dB]. ECA/ECX NMR Training Guide, v4.3-a 23

Figure 24 1 H Selective 90 pulse width ECLIPSE+ NMR Training Guide, v4.3.4 24

1 H Spin-lock 90 Purpose: Calibrate the spin-lock 90 pulse and spin lock attenuator. Sample needed: 5% CHCl 3 in acetone d-6 doped with trace Cr(acac) 3 Instrument preparation: A well-shimmed system with the probe tuned to the sample ( 1 H). Some experiments using this pulse: tocsy_pfg_s.exp Experiments used in this calibration: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/service/spin_lock90_check.exp A proton survey spectrum needs to be run first. Run the survey spectrum using single_pulse.exp. Transfer both the receiver gain value noted from the Delta Console and the on resonance condition for the CHCl 3 peak to the appropriate fields in spin_lock90_check.exp. Transfer the on resonance condition to x_offset. Set x_pulse to twice what you feel is suitable for covering the desired bandwidth. Recall that the B 1 field can be calculated by: B 1 (Hz) = 1/(4*pulse width [s]). Thus, for a 4 khz field on a 300MHz spectrometer, you would need a pulse width of about 62.5 µs. Array obs_attenuator from 15-30[dB] in steps of 1[dB]. When the data set returns, process it as in the 1 H obs 90 pulse width calibration. Look for the best null. Transfer the attenuator value that gave the best null to obs_attenuator. In the Header section of spin_lock90_check.exp, click on Header. This will bring up a window allowing you to change the flags in the Header section. Click on auto_dwell. Then click on Add and then Done. The experiment window should recompile and display the auto_dwell flag. Make this flag false by un-checking the box. Now, array x_pulse centered around twice the desired pulse width value. When the arrayed data set returns, process it as in the 1 H obs 90 pulse width calibration (Figure 25). Divide the null value by two to get your pulse width. Note the pulse width and attenuator value. Figures 26 and 27 illustrate where to change these values in the graphical tool. ECA/ECX NMR Training Guide, v4.3-a 25

Figure 25 Spin-Lock 90 pulse width * Figure 26 Change the highlighted values to save the Spin-Lock 90 pulse width * Figure 27 Change the highlighted values to save the Spin-Lock 90 attenuator ECLIPSE+ NMR Training Guide, v4.3.4 26

19 F Obs 90 Purpose: Determine the 19 F observe 90 pulse width Sample needed: 0.05% trifluorotoluene in benzene-d6 Instrument preparation: Shim and tune probe to the sample ( 19 F). Experiment used: /usr/delta/global/experiments/service/obs90_check.exp /usr/delta/global/experiments/service/obs90_array.exp Process list used: /usr/delta/global/process_lists/std_proton.list A survey spectrum needs to be collected first to get the exact position of the fluorine peak. Set x_domain to Fluorine19. Set recvr_gain to an appropriate value. Set x_offset to 62[ppm] and x_sweep to 100[ppm]. Submit the experiment. Copy the x-position of the 19 F peak and paste it into x_offset in obs_90_array.exp. Set relaxation_delay to 15[s]. Submit the experiment. Process the data and find your best null. Divide the null by two to get the 19 F obs 90. Figure 28 shows a typical result. Figure 29 highlights where to save your pulse width in Machine Config?. Figure 28 19 F Obs 90 pulse width ECA/ECX NMR Training Guide, v4.3-a 27

* Figure 29 Change the highlighted value to save the 19 F Obs 90 pulse width ECLIPSE+ NMR Training Guide, v4.3.4 28

31 P Obs 90 Purpose: Determine the 31 P observe 90 pulse width Sample needed: 1% trimethylphosphite in acetone-d6 Instrument preparation: Shim and tune probe to the sample ( 31 P, 1 H) Experiment used: /usr/delta/global/experiments/service/single_pulse_dec.exp Process list used: /usr/delta/global/process_lists/std_carbon.list A survey spectrum needs to be collected first to get the exact position of the phosphorus peak. Set x_domain to Phosphorus31. Set recvr_gain to an appropriate value. Set x_offset to 140[ppm] and x_sweep to 200[ppm]. Set scans to 1, x_prescans to 0 and x_angle to 90[deg]. Submit the experiment. Copy the x-position of the 31 P peak and paste it into x_offset. Array x_90_width from 2-50[us] in 2[us] steps. Set relaxation_delay to 30[s]. Submit the experiment. Process the data and find your best null. Divide the null by four to get the 31P obs 90. Figure 30 shows a typical result. Figure 31 highlights where to save your pulse width in Machine Config?. Figure 30 31 P Obs 90 pulse width ECA/ECX NMR Training Guide, v4.3-a 29

* Figure 31 Change the highlighted values to save the 31 P Obs 90 pulse width ECLIPSE+ NMR Training Guide, v4.3.4 30

15 N Obs 90 Purpose: Determine the 15 N observe 90 pulse width Sample needed: 90% formamide in dmso-d6 Instrument preparation: Shim and tune probe to the sample ( 15 N, 1 H) Experiment used: /usr/delta/global/experiments/service/single_pulse_dec.exp Process list used: /usr/delta/global/process_lists/std_carbon.list A survey spectrum needs to be collected first to get the exact position of the nitrogen peak. Set x_domain to Nitrogen15. Set recvr_gain to an appropriate value. Set x_offset to 108[ppm] and x_sweep to 200[ppm]. Set scans to 1, x_prescans to 0 and x_angle to 90[deg]. Submit the experiment. Copy the x-position of the 15 N peak and paste it into x_offset. Array x_90_width from 2-50[us] in 2[us] steps. Set relaxation_delay to 160[s]. Submit the experiment. Process the data and find your best null. Divide the null by two to get the 15 N obs 90. Figure 32 shows a typical result. Figure 33 highlights where to save your pulse width Machine Config?. Figure 32 15 N Obs 90 pulse width ECA/ECX NMR Training Guide, v4.3-a 31

* Figure 33 Change the highlighted values to save the 15 N Obs 90 pulse width ECLIPSE+ NMR Training Guide, v4.3.4 32

III. Commonly Used Experiments in Structure Elucidations The following sections describe how to perform some commonly used NMR experiments using the DELTA software. All the data in these descriptions were collected on a sample of quinidine in CDCl 3. A numbering scheme and structure for quinidine is shown in Figure 34. All of the subsequent discussions and figures will refer to this scheme. H 3 7 C O 8 9 H 12 OH H 14 H 14' 6 5 4 3 10 N 11 1 2 13 17 N 18 16 19 15 20 21 22 CH 2 C 20 H 24 N 2 O 2 Figure 34 Structure and numbering scheme for quinidine ECA/ECX NMR Training Guide, v4.3-a 33

1D Proton Purpose: Collect a 1D proton data set Experiment(s): /usr/delta/global/experiments/single_pulse.exp Pulse width(s) used: 1 H Obs 90 Processing List(s): /usr/delta/global/process_lists/std_proton_autophase.list A 1D proton NMR spectrum is normally the first experiment run on a sample. A proton spectrum yields a wealth of information including: total number of protons present (from integrations), the basic types of protons present (from chemical shift) and proton connectivities (from coupling constants and splitting patterns). Parameters to set Check auto_gain to automatically find the best receiver gain value. A correctly set receiver gain will avoid clipping the data. Set scans to the number desired. Processing Load or assemble an appropriate processing list and then peak-pick and integrate the data. Loading a Processing List In 1D Processor, it is possible to build processing lists from the various menus or to load a list from disk. Click, which is near the button, to open a standard list from disk. The box in Figure 35 will appear. Click to change to the global directory. Choose std_proton_autophase.list and then click OK. A processing list will appear. Click Process to execute the processing list. Touch up the phase with P0 and P1 if necessary. Alternatively, one can load a proton processing list by clicking on, located in the row of macro icons in 1D Processor. ECLIPSE+ NMR Training Guide, v4.3.4 34

Figure 35 Choosing a process list from disk, the global button is indicated by the red box Using the Pointer Tool to Manipulate Data To manipulate data graphically, the Pointer Tool appears on both 1D and 2D data. The tool is divided into 13 modes. By default, Zoom mode appears the first time you open a 1D Processor. Every time thereafter, the last-used tool will appear in data windows. By clicking where it says Zoom with the left-mouse button, you will be able to select any of the other remaining major modes. Using these modes, one can peak pick, integrate and Figure 36 The Zoom mode of the Cursor Tool Figure 37 The fourteen available modes in the Cursor Tool ECA/ECX NMR Training Guide, v4.3-a 35

add text annotation, to name but a few functions. Each of the major modes has unique features, including hot key functions, which are available only when a particular major mode is selected. To view the hot keys available for the presently selected mode, select. Figure 38 shows the available hot keys for Zoom. Moreover, there are three Zoom Figure 38 The hot keys available from the Zoom cursor hot keys which are particularly useful. These three hot keys are: Home, which resets both axes to the original limits, Backspace, which will step back to previous zooms, one zoom at a time, and End, which will scale the data in the y-dimension to fill the screen. Finally, it should be noted that from whatever major mode you are in, one can always get Zoom data by holding down the Shift key. Using the Peak Mode of the Pointer Tool The automatic peak picking in a 1D data set is done with regards to peak pick thresholds. There are three threshold lines drawn on data. The positive peak threshold is drawn in green, while the noise threshold and negative peak threshold are drawn in gray and red, respectively. The Peak mode of the pointer tool contains tools to adjust these thresholds. Since there are many buttons in this mode, it may initially be difficult to remember what the icons mean. One can view what all the buttons in any particular major mode correspond to by clicking the middle mouse button on the name of the tool (i.e. in this case, middle-click where it says Peak). It should be noted that most of the NMR systems are shipped with mice that have a wheel for the middle mouse button. Pressing on the wheel is equivalent to pressing a middle mouse button. If you click the middle mouse button in Peak mode, you will see the menu in Figure 39. It is possible to select the sub-modes within this menu using the mouse. To adjust the positive peak-picking threshold, choose Adjust peak threshold. Move the green line so that the tops of all the desired peaks are above the threshold. Click ECLIPSE+ NMR Training Guide, v4.3.4 36, which is located in the top row of

Figure 39 The sub-modes seen in Peak by pressing the middle mouse button icons, to peak pick the data set. An example showing the peak pick threshold is given in Figure 40.The threshold can be re-positioned if desired but the peak picks will not be repicked until you click again. With peak-picked data, it is possible to use one of the Peak mode hot keys to help elucidate coupling constants. Select two peaks of interest with Select more peaks. Now hit the letter j on the keyboard. The frequency difference between these peaks, in Hz, will be displayed on the data. Additionally, a new peak-pick will appear at the center of the two selected peaks. This centroid peak can be used to identify a peak position or for nesting additional coupling information. To generate additional J s be sure to unselect the currently selected peaks with Unselect peaks or by hitting the letter u on the keyboard. The difference algorithm will always give the frequency difference between the two outer-most selected peaks. Figure 41 shows an example of the J Coupling feature. Positive peak threshold line Figure 40 Setting the positive peak threshold in Peak mode ECA/ECX NMR Training Guide, v4.3-a 37

Type the letter J 2 5 O 9 6 C H 3 5 OH H N 1 N 2 CH 2 1 6 9 Figure 41 The result of the J Coupling feature in Peak mode (see values above the data) Using the Integral Mode of the Pointer Tool The integration in a 1D data set can be done automatically by clicking (located in the top row of icons of 1D Processor). However, it is common to integrate data manually. To integrate data manually, choose, which is Create integral, while in Integral mode. Remember to middle-click on the name of the tool (in this case Integral) if you are unsure of the identity of a particular sub-mode s icon. An integral is created by choosing a start and stop point about a peak or group of peaks. The start and stop points are indicated by two small blue boxes drawn on the data. To generate a start and stop point, single-click-hold the left mouse button in the gray area underneath the x-axis of the data (Figure 42). You will see a blue box drawn on the data. Drag across to the desired stopping point and then release the mouse button. Notice that by keeping the mouse focus in the gray area below the x-axis forces the blue boxes to be drawn on data points (i.e. along the baseline). Therefore, drawing integrals while in the gray area is the best way to get nicely drawn integrals. Should an integral need to be adjusted, use the sub-modes Change slope of integral or Adjust integral. ECLIPSE+ NMR Training Guide, v4.3.4 38

Click-hold-drag Put Mouse Focus in Here When Drawing Integrals Plotting Data Figure 42 Be sure to stay in the gray area when drawing an integral with Create integral To plot a data set click, located in the top row of icons. The resulting hardcopy will be WYSIWYG (what-you-see-is-what-you-get). Assignment Strategy In quinidine, we know that there are 24 protons, therefore, we must integrate at least this many protons in our proton spectrum to have a correct structure. If there are additional protons, one might look for solvent or starting material resonances. With some prior basic chemical shift information, one can confirm that there are correct numbers of aromatic and aliphatic protons, and that the alcohol, methoxy and vinyl protons are present. Note that it is possible for exchangeable protons, such as alcohol protons, to be absent in a proton spectrum. If such protons are present, they may not integrate to an integer value. Some obvious assignments are given in Figure 43. ECA/ECX NMR Training Guide, v4.3-a 39

2 22 7 21 12 OH Figure 43 A 1D proton spectrum of quinidine ECLIPSE+ NMR Training Guide, v4.3.4 40

1D Carbon and DEPT 135 Purpose: Identify the number and types of carbons present Experiments: /usr/delta/global/experiments/single_pulse_dec.exp and dept.exp Pulse width(s) used: 13 C Obs 90, 1 H Decoupling 90 Processing List(s): /usr/delta/global/process_lists/std_carbon_autophase.list A 1D carbon is normally run on organic samples. A carbon spectrum gives information about the number and type of carbons (from chemical shift) that are present in the sample. To confirm the types of carbons present, one can also run a DEPT 135 (Distortionless Enhancement by Polarization Transfer) experiment. The DEPT experiment yields different phases for CH s and CH 3 s versus CH 2 s, which allows for their discrimination. Also, because quaternary carbons do not appear in a DEPT, signals that may be hidden under residual solvent resonances are readily revealed. Finally, because the DEPT experiment utilizes a polarization transfer to increase signal intensity, it usually requires fewer scans than a carbon to achieve a comparable signal-to-noise. Parameters to set Check auto_gain in both experiments to automatically find the best receiver gain values. A correctly set receiver gain will avoid clipping the data. Set scans to the number desired. In the DEPT experiment, set selection_angle to 135[deg]. Additionally, to maximize available NMR time, these experiments can be set to run to a particular signalto-noise value. To do this, enter a non-zero value into sn_ratio. A value of zero turns this feature off. By default, the algorithm looks for the tallest peak, which is not a residual lock solvent peak, and uses its intensity in the signal-to-noise calculation. However, it is also possible to specify both signal and noise regions. Specifying a signal region can be very useful if one is looking to run the experiment until a carbonyl carbon appears. The signal region can be set to look for the carbonyl peak in an anticipated region. On the Header tab of the experiment window, click. A box similar to Figure 44 should appear. Click on sn_signal_start, Add, sn_signal_stop, Add, and then Done. Enter a region where you expect to find the peak of interest (Figure 45). Now when you submit the experiment, it will run the experiment until it reaches sn_ratio for the peak in the specified region or scans, whichever comes first. If desired one can use the same procedure to specify a noise region. ECA/ECX NMR Training Guide, v4.3-a 41

Figure 44 Adding the signal flags Figure 45 Adjust these parameters to a suitable region, units must be specified. Processing After the data have been transformed, phase the carbon data, and peak pick as usual. For the DEPT 135, phase the data so that the methyl and methine peaks are up and the methylene peaks are down. Make sure that if any manual phasing was done to click and to re-process the data. Otherwise, the phase changes will not be transferred to Data Slate. Drag both data sets to a Data Slate and chose View -> Connect X to connect the x axes. With the axes connected, zooms will track identically for both data sets. Assignment Strategy In quinidine, we know that there are 20 distinct carbons. Therefore, we must count at least this many carbons in our 1D carbon spectrum to have a correct structure. Remember that chemically equivalent carbons will show up as one peak, so the number of carbons in the molecule may not equal the number of carbons counted in the spectrum. If there are additional carbons, one might look for solvent or starting material resonances. In DEPT135 spectra, both CH 3 s and CH s are usually phased up while CH 2 s are phased down. Thus, the DEPT135 data in Figure 46 shows that are 11 methyls/methines and 5 methylenes present. Moreover, since quaternary carbons do not appear in DEPT spectra, the 4 carbons that appear in the 1D carbon, but are absent in the DEPT 135, are assigned as quaternary carbons. Thus, all the types of carbons expected are present. The DEPT experiments prove particularly useful for discerning a methine carbon from a methylene ECLIPSE+ NMR Training Guide, v4.3.4 42

carbon when chemical shift information is ambiguous and for seeing carbons buried under residual solvent peaks. CH CH CH CH CH CH CH CH CH CH CH CH CH 2 CH 2 CH 2 CH 2 2 22 7 q q q q Figure 46 1D carbon and DEPT135 result indicating types or carbons ECA/ECX NMR Training Guide, v4.3-a 43

DQF-COSY Purpose: Elucidate proton connectivities Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/ecp_grad/single_shape/dqf_cosy_pfg_s.exp Pulse width(s) used: 1 H Obs 90 Processing List(s): /usr/delta/global/process_lists/2d_cosy_abs.list A DQF-COSY (Double Quantum Filtered) experiment is run to assign proton connectivities. The double quantum filter attenuates singlets such as a water peak or methoxy peak. Suppression of these types of signals allows one to set a higher receiver gain than that used in its corresponding proton experiment. Parameters to set Run a 1D proton NMR. Note the gain value determined for the proton spectrum. As a first guess, use this receiver gain in the DQF-COSY experiment. Remember that the double quantum filter attenuates large singlets so if these types of signals are present, the receiver gain can be set to a higher value. Zoom the peaks in the proton data so there is about 0.5[ppm] on either side of the peaks. In the DQF-COSY experiment window, click and then. Now click on the zoomed proton data set. The x_offset and x_sweep will change to reflect the zoomed data set. Turn the spinner off. Loading High-Resolution Projections in Homonuclear Data In the nd Processor, click to send the data to the 2D Viewer. A window resembling Figure 47 will result. Notice that there are low-resolution 1D projections drawn on the data. These projections are generated from the intensity dimension of the 2D data. It is common to load high-resolution data (i.e. your proton data) into these axes. Click in the 2D Viewer to toggle operations from disk to finger mode. Select Display -> High- Res -> Load X Projection, and then click on the processed proton spectrum. This will load the proton data set into the x-axis of the viewer. Repeat the procedure using Load Y Projection. Notice now that the data contained along the axes have real filenames. Upon loading the high-resolution projections, the 1D and 2D data will be automatically connected. Therefore, any zooming done in either the 1D or 2D portions of the window will occur in concert. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. ECLIPSE+ NMR Training Guide, v4.3.4 44

Figure 47 Loading high-resolution projections Assignment Strategy To assign COSY peaks, look for matching cross-peaks off the diagonal. Figure 48 shows an expansion of the aromatic region of a DQF-COSY spectrum with the coupled aromatic protons traced out and a structure highlighting these connections. Figure 49 shows the full spectrum. Note that in the full spectrum, the data is symmetric about the diagonal drawn. ECA/ECX NMR Training Guide, v4.3-a 45

2 5 1 6 9 C H 3 O 9 6 5 H N 1 2 OH N CH 2 Figure 48 Expansion of the aromatic region ECLIPSE+ NMR Training Guide, v4.3.4 46

Figure 49 A DQF-COSY spectrum shown with a diagonal ECA/ECX NMR Training Guide, v4.3-a 47

HMQC Purpose: Elucidate proton-carbon connectivities Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/single_pulse_dec.exp /usr/delta/global/experiments/ecp_grad/single_shape/hmqc_irr_pfg_s.exp Pulse width(s) used: 1 H Obs 90, 13 C Decoupling 90 Processing List(s): /usr/delta/global/process_lists/2d_inverse_abs.list An HMQC (Heteronuclear Multiple Quantum Coherence) experiment is run to assign proton-carbon connectivities. The HMQC is known as an inverse detected experiment because it detects the more sensitive 1 H nucleus instead of 13 C. Thus, this experiment is especially useful if you are sample limited because it will probably require fewer transients. Moreover, because the experiment filters out 12 C- 1 H peaks, the receiver gain should be set higher than that used to collect a corresponding proton data set to take advantage of the inverse detection. Parameters to set Run 1D proton and 1D carbon spectra. Set recvr_gain to a sensible value. Remember that because this experiment suppresses 12 C- 1 H signals, you will need to use a value higher than used for the 1D proton experiment. Optimize x_offset and x_sweep from the processed 1D proton data. Do the same for y_offset and y_sweep from the processed 1D carbon data. Turn the spinner off. Loading High-Resolution Projections in Heteronuclear Data In the nd Processor, click to send the data to the 2D Viewer. Click in the 2D Viewer to toggle operations from disk to finger mode. Select Display -> High-Res -> Load X Projection, and then click on the processed 1D proton spectrum. This will load the proton data set into the x-axis of the viewer. Repeat the procedure using Load Y Projection but click instead on the 1D carbon spectrum. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. Assignment Strategy The spots seen on an HMQC spectrum can be traced directly to the corresponding 1H and 13C peaks in the high-resolution projections. These correlations show the protons and carbons that are directly attached to one another. Figure 50 shows an HMQC data set with correlations for both 12 and 22 traced out. Additionally, a structure highlighting the connections is also shown. ECLIPSE+ NMR Training Guide, v4.3.4 48

22 12 C H 3 O OH H N N H H 2 2 Figure 50 An HMQC Spectrum ECA/ECX NMR Training Guide, v4.3-a 49

HETCOR Purpose: Elucidate proton-carbon connectivities Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/single_pulse_dec.exp /usr/delta/global/experiments/chshf.exp Pulse width(s) used: 13 C Obs 90, 1 H Decoupling 90 Processing List(s): /usr/delta/global/process_lists/2d_ch_abs.list An HETCOR (Heteronuclear Correlation) experiment is run to assign proton-carbon connectivities. In contrast to the HMQC, a HETCOR detects 13 C. Thus, this experiment will usually require more scans than an HMQC. However, if you are not sample limited, the HETCOR has the advantage of good resolution in the carbon domain. Note that chshf is the HETCOR experiment. Parameters to set Run 1D proton and 1D carbon spectra. Click auto_gain to automatically determine a receiver gain value. Optimize x_offset and x_sweep from the processed 1D carbon data. Do the same for y_offset and y_sweep from the processed 1D proton data. Turn the spinner off. Processing In the nd Processor, click to send the data to the 2D Viewer. Click in the 2D Viewer to toggle operations from disk to finger mode. Select Display -> High-Res -> Load X Projection, and then click on the processed 1D carbon spectrum. This will load the carbon data set into the x-axis of the viewer. Repeat the procedure using Load Y Projection but click on the 1D proton data. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. Assignment Strategy The spots seen on a HETCOR spectrum can be traced directly to the corresponding 1H and 13C peaks in the high-resolution projections. These correlations show the protons and carbons that are directly attached to one another. Figure 51 shows a HETCOR data set with correlations for both 12 and 22 traced out. Additionally, a structure highlighting the connections is also shown. Note that the axes on the data set are the reverse of the HMQC. This difference in axes is not just cosmetic; it can also affect the ultimate data resolution in x and y. Figure 52 shows an overlay of HETCOR (red) and HMQC (green) data. Note that to enable the overlay of these data an extra Transpose has to be added to the y-processing list of one of the data sets. In Figure 52, the HETCOR data more clearly shows that the two protons buried under the resonance at ~2.88[ppm] are on different carbons. The reason that the carbon resolution may be better in the HETCOR than in the HMQC is because you are dividing the wider carbon spectral width by more data points ECLIPSE+ NMR Training Guide, v4.3.4 50

in the carbon-detected experiment. This is because the directly detected domain always contains more data points. This does not mean that HETCOR should always be preferred over inverse-type experiments, but rather that one has to be aware of setting the conditions 12 22 C H 3 O H 12OH N N H H 22 Figure 51 A HETCOR spectrum, the box corresponds to the expansion in Figure 52 ECA/ECX NMR Training Guide, v4.3-a 51

Figure 52 Expansion comparing HETCOR and HMQC ECLIPSE+ NMR Training Guide, v4.3.4 52

Edited HSQC Purpose: Elucidate proton-carbon connectivities and multiplicity of carbons Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/single_pulse_dec.exp /usr/delta/global/experiments/multiplicity_hsqc_pfg_s_phase_pn.exp Pulse width(s) used: 1 H Obs 90, 13 C Decoupling 90 Processing List(s): /usr/delta/global/process_lists/2d_inverse_phase.list An HSQC (Heteronuclear Single Quantum Coherence) spectrum is run to assign protoncarbon connectivities. The HSQC is known as an inverse detected experiment because it detects the more sensitive 1 H instead of 13 C. The phase sensitive version shown here also affords information similar to a DEPT 135, in that one can identify the multiplicities of the carbons from the peak phase. Parameters to set Run 1D proton and 1D carbon spectra. Set recvr_gain to a sensible value. Remember that because this experiment suppresses 12 C- 1 H signals, you will need to use a value higher than used for the 1D proton experiment. Optimize x_offset and x_sweep from the processed 1D proton data. Do the same for y_offset and y_sweep from the processed 1D carbon data. Turn the spinner off. Phasing 2D Data In the nd Processor, click to send the data to the 2D Phaser (if you are processing an FID, be sure to load in a processing list first!). Using Create cursor, drop some cursors along the data, on the peaks you wish to phase. The cursors will mark where the data will be sliced (Figure 53). Click. The window will now show you the slices you selected. Phase the data with the p0 and p1 buttons provided (Figure 54). You may find it easier to phase one dimension at a time. That is, phase one axis, apply the phase and then drop cursors again to phase the next dimension. Click to apply any phase changes. If you are happy with the phasing click to send the data to the 2D Viewer. Load the high-resolution projections. You need to load the 1D proton with Load X Projection and the 1D carbon with Load Y Projection. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. ECA/ECX NMR Training Guide, v4.3-a 53

Figure 53 Dropping cursors in the 2D Phaser Figure 54 Data after phasing in the 2D Phaser ECLIPSE+ NMR Training Guide, v4.3.4 54

Assignment Strategy The spots seen on an HSQC spectrum can be traced directly to the corresponding 1H and 13C peaks in the high-resolution projections. These correlations show the protons and carbons that are directly attached to one another. Additionally, in this Edited HSQC spectrum, the phase affords multiplicity information analogous to the DEPT 135 experiment. In Figure 55, the contours have been colored green and red, marking the positive and negative contours, respectively. The positive contours correspond to methyl and methine carbons, whereas the negative contours correspond to methylene carbons. In the figure, the correlations for both 12 and 22 are traced out. Additionally, a structure highlighting the connections is also shown. Note also that in the figure, both the 1D carbon and DEPT 135 data have been loaded on the y-axis for emphasis. To accomplish this, the carbon was loaded into the high-resolution projection while the DEPT135 was loaded into the high-resolution slice. 12 22 C H 3 O H 12 OH N N H 22 H Recolor data to show CH, CH 3 versus CH 2 Figure 55 Edited HSQC spectrum ECA/ECX NMR Training Guide, v4.3-a 55

1H-15N Correlations with HMQC Purpose: Elucidate proton-nitrogen connectivities Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/ecp_grad/single_shape/hmqc_irr_pfg_s.exp Pulsewidth(s) used: 1 H Obs 90, 15 N Obs 90, 15 N Decoupling 90 Processing List(s): /usr/delta/global/process_lists/2d_hmbc_abs.list An HMQC spectrum is usually run to assign proton-carbon connectivities. However, by choosing a different j_constant and changing the y_domain to nitrogen, on can get a 1 H- 15 N correlation. This inverse experiment is especially useful because it observes the much more sensitive proton nucleus instead of nitrogen, which in practice is hard to observe directly. Detecting the nitrogen indirectly then affords a great time savings over a direct observe 1D nitrogen experiment. Additionally, the experiment gives long-range 1 H- 15 N correlations that can be used to unambiguously assign the nitrogens without consulting a chemical shift table. Parameters to set The recvr_gain will need to be set to a large value. Optimize x_offset and x_sweep from the 1D proton data. Set y_offset and y_sweep to a range appropriate for your compound. You may need to consult a chemical shift table to set this range. Set scans. Change y_domain to Nitrogen15. Note that the correct pulse widths and gradient ratios are parsed into the experiment window automatically when you select the 15 N domain. If you do not have an auto tune probe make sure you check the box for force_tune in the top of the experiment window. You will be prompted to tune the LF and HF channel by hand after you submit the experiment. Set j_constant to a value appropriate for your compound. In this example, a 2,3 J constant of 6[Hz] was used. Turn the spinner off. Processing Click to send the data to the 2D Viewer. Note that a Notch Filter was added to the processing list in X to take out the methoxy ridge. Load the 1D proton data with Load X Projection. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. Assignment Strategy One can read off the correlations exactly as in an HMQC or HSQC data set. The only difference is that in this data, the positions of the nitrogen peaks are read off from the low-resolution y-projection and not from a high-resolution 1D data set. Figure 56 shows that two different nitrogens are seen as was expected from the structure. A structure highlighting the correlations is also shown in the figure. Note that the correlations seen in this type of experiment are highly dependent on the magnitude of the j_constant used. ECLIPSE+ NMR Training Guide, v4.3.4 56

12 13 19 17 14 2 5 1 H 3 C O 5 N OH 12 14 1 17 N 1 2 19 CH 2 N 18 N 3 Figure 56 1H-15N HMQC spectrum ECA/ECX NMR Training Guide, v4.3-a 57

HMBC Purpose: Elucidate long-range proton-carbon connectivities Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/single_pulse_dec.exp /usr/delta/global/experiments/ecp_grad/single_shape/hmbc_pfg_s.exp Pulsewidth(s) used: 1 H Obs 90, 13 C Obs 90 Processing List(s): /usr/delta/global/process_lists/2d_hmbc_abs.list The HMBC (Heteronuclear Multiple Bond Correlation) experiment is designed to give long-range 2,3 J(C,H) proton-carbon connectivities. An HMBC data set is useful for assigning quaternary carbons and as a check that other correlations have been assigned correctly. Parameters to set Set the recvr_gain to a sensible value. Remember that because this experiment suppresses 12 C- 1 H signals, you will need to use a value higher than that used for the correponding 1D proton experiment. Optimize x_offset and x_sweep from the processed 1D proton data. Do the same for y_offset and y_sweep from the processed 1D carbon data. Turn the spinner off. The default setting for long_range_j can be modified if needed. Processing Click to send the data to the 2D Viewer. Load the 1D proton data with Load X Projection. Load the 1D carbon data with Load Y Projection. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. Assignment Strategy An HMBC spectrum provides a wealth of information. One can read off the correlations exactly as in an HSQC or HMQC data set. However, an HMBC will usually give you many more correlations. Figure 57 shows an HMBC data set. Figure 58 shows one strip of the data at H12 and also shows a structure highlighting the correlations. Note especially that correlations to quaternary carbons 10, 11 and 13 are seen. The HMBC pulse sequence is designed to give long-range correlations and suppress single bond correlations. However, if single bond correlations do break through, they appear as doublets. Some breakthrough correlations are indicated in Figure 59. This figure has the HMBC data (green) overlaid on top of HSQC data (red). ECLIPSE+ NMR Training Guide, v4.3.4 58

12 14 13 1 10 11 Figure 57 An HMBC spectrum, the box corresponds to the expansion in Figure 59 H12 14 13 H 3 C O H 12 OH 1 10 11 4 13 N 1 14 1 10 N CH 2 11 Figure 58 HMBC data show long-range ( 2,3 J) correlations ECA/ECX NMR Training Guide, v4.3-a 59

Figure 59 Overlay of HSQC (red) and HMBC (green), break-through single bond correlations are circled ECLIPSE+ NMR Training Guide, v4.3.4 60

1D DPFGSE-NOE Purpose: Elucidate through-space interactions Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/dpfgse_noe_pfg_s.exp Pulsewidth(s) used: 1 H Obs 90, 1 H Selective 90 Processing List(s): /usr/delta/global/process_lists/std_proton_autophase.list The Double Pulsed Field Gradient Spin Echo experiment has largely supplanted the traditional difference NOE experiment. This is because it does not produce artifacts caused by subtraction errors or Bloch-Siegert shifts. Note that this experiment contains a mix_time. This is because unlike the traditional steady-state difference NOE experiment, the DPFGSE-NOE experiment produces a transient NOE. The intensity of the enhancements seen in this experiment is highly dependent on the duration of the mix time. Parameters to set Run a 1D proton experiment first. You will need to set recvr_gain to a value higher than that used in the 1D proton experiment. Using Copy position to Buffer from the Pick cursor, select the peak you wish to irradiate from the proton data set. Move the mouse into the x_offset field, double-click the right mouse button, and then click the middle mouse button to paste in the offset value. Set mix_time to a sensible value. Turn the spinner off. Processing Process the 1D proton spectrum as usual. Process and phase the DPFGSE-NOE spectrum so that the irradiated peak is negative. It is helpful to move both the 1D proton and DPFGSE-NOE spectrum to a Data Slate and to connect their x-axes. Assignment Strategy There are two basic types of structural information gained from NMR data. One type probes through-bond interactions while the other type probes through space-interactions. The NOE experiments like the 1D DPFGSE-NOE experiment probe through-space interactions. The NOE effect can be quite weak and falls off precipitously with distance 1 (~ r 6 where r is inter-atomic distance). Since one is investigating through-space interactions, it is very useful to make a model to use when deciding which protons to irradiate and when trying to interpret results. This experiment is especially useful for assigning diastereotopic protons. In the example, the peaks thought to be 14 and 14' were irradiated. Since the peak irriadiated at ~2.1[ppm] shows an enhancement to 21 and the ECA/ECX NMR Training Guide, v4.3-a 61

peak irradiated at 1.16 [ppm] does not, the peak at ~2.1[ppm] must be 14'. This is because only the proton closer to 21 should show a correlation (see Figure 60). C H 3 O H OH N N H 14 14 H 21 H CH 2 * irradiated at 1.14 ppm irradiated at 2.00ppm proton spectrum 21 no enhancement * 14" * 14 Figure 60 A 1D NOE-DPFGSE experiment, the * s indicate where peaks were irradiated ECLIPSE+ NMR Training Guide, v4.3.4 62

ROESY Purpose: Elucidate through-space interactions Experiments: /usr/delta/global/experiments/single_pulse.exp /usr/delta/global/experiments/roesy/t_roesy_phase.exp Pulsewidth(s) used: 1 H Obs 90 Processing List(s): /usr/delta/global/process_lists/2d_homo2d_phase.list It is often useful to run a two-dimensional NOE experiment. This is because one can obtain correlations for all protons in one experiment. The ROESY (Rotating frame Overhauser Effect Spectroscopy) is a good candidate for small molecules. This is because for compounds with molar masses ~1000 3000, cross signals can disappear due to the fact that the NOE effect modulates as a function of molecular correlation time. Parameters to set Run a 1D proton first. Use the receiver_gain for the ROESY experiment. Optimize x_offset and x_sweep from the processed 1D proton data. Set x_spinlock_atn so that the spinlock_strength is sufficient to cover the desired bandwidth. Set an appropriate mix_time. It is often useful to run several experiments with slightly different mix times. Turn the spinner off. Processing Click to send the data to the 2D Phaser. Phase the data so that the cross-peaks are negative and the diagonal peaks are positive. Click to send the data to the 2D Viewer. Load the 1D proton data with Load X Projection. Contour the data, as needed, using the Level Tool. See Appendix D for a detailed explanation of the Level Tool. Assignment Strategy As in the 1D NOE experiment, it is very useful to refer to a model of your compound when trying to make assignments. The NOE correlations will appear with a phase opposite to that of the diagonal. In Figure 61, the NOE correlations are shown in red, while the diagonal is shown in green. An expansion of the aliphatic region of the spectrum is shown in Figure 62. ECA/ECX NMR Training Guide, v4.3-a 63

Figure 61 A ROESY spectrum ECLIPSE+ NMR Training Guide, v4.3.4 64

Figure 62 An expansion of the ROESY spectrum ECA/ECX NMR Training Guide, v4.3-a 65

IV. Using Preferences to Customize an Account There are a number of preferences that can be set to suit a user's taste. These preferences include whether or not to display certain features (i.e. peak pick, integrals, grid), default file directories, printer options etc. The default values for all such items are contained in a text-based file in your home directory on the workstation computer. The advised mode of altering this file is with the GUI Preferences tool. In the Delta Console, select File- >Preferences After a few moments, the Preferences tool will appear (Figure 63). At the top of this tool are twelve icons that select the different "pages" within the tool. Because there are a large number of Preferences, it is usually best to find an individual preference using the Options-> Search function within this tool. In Figure 64, a search was performed on integrals. If a preference is selected in the Search Tool, the Preferences tool will change to the appropriate page and be highlighted in yellow (Figure 63). Figure 63 Preferences? tool found in the Delta Console Figure 64 Search Tool contained in the Preferences? Tool ECLIPSE+ NMR Training Guide, v4.3.4 66

V. File Management Setting default paths with the Preferences Tool The default paths for data, as well as all other default paths, can be modified using the Preferences tool. The paths are available on the Directory tab. Click to access this Figure 65 The Directory tab Tab (Figure 65). Keep in mind that the directory you choose must already exist. One can browse currently available directories by clicking. Using File Manager In addition to the operating system s file management tools, Delta provides the File Manager. This tool provides additional functionality specific to NMR data files. To start the tool, click from the Master Console. The tool in Figure 66 should appear. ECA/ECX NMR Training Guide, v4.3-a 67

Figure 66 File Manager tool Deleting Processed Data with File Manager It is common to keep raw NMR data (i.e. the FID) and to discard processed NMR data. The File Manager provides a convenient way of doing this. The first step is to move to the directory containing your data. Click on Select Directory and choose Data. Click the filename on which to operate. With this filename highlighted, choose Data -> Purge Processed. The Purge Options box should appear (Figure 67). This window allows you to purge the currently selected files, files in the current directory or all the files in all the user's subdirectories. The only extensions remaining after this action will have units of [s]. ECLIPSE+ NMR Training Guide, v4.3.4 Figure 67 Purge file options 68

Performing a File Search with File Manager The File Manager has the capability of performing very detailed searches for NMR files. To initiate a search in the File Manger, as well as in other Delta tools, click. The window in Figure 68 should appear. Click any desired boxes and adjust the Booleans as necessary. Click to initiate the search. The example in the figure searches Figure 68 Performing a search for NMR data files for all FID with a mix_time of 0.5[s]. The result of this search is shown in Figure 69. To view a file, select it with the mouse and then click on the desired viewer at the bottom of the tool. Alternatively, one can drag files out to the desktop with right-mouse-button drag and drop. ECA/ECX NMR Training Guide, v4.3-a 69

Comparing Files with File Information Figure 69 Click on a viewer or drag file onto the desktop In addition to searching on parameters, it is also possible in Delta to directly compare two data files parameters. Click Master Console ->, which is the open data icon. Choose a filename and extension and then click. A new window will display the parameters for that file. You can change to All to see all the parameters. To compare another data sets parameters, choose File -> Compare. Select another filename and extension. The second file and its parameters will then be displayed in the same window (Figure 70). Any parameters common to the two data files will be displayed in black. Parameters common to the two data files, which have different values, will be displayed in red. Parameters that are unique to a data file will be displayed in green. ECLIPSE+ NMR Training Guide, v4.3.4 70

ECA/ECX NMR Training Guide, v4.3-a Figure 70 Comparing two files using File Information VI. Shutting Down/Starting up Your System Eventually it will be necessary for you to shut down and restart your system. For example, you might learn that your physical plant is scheduling a power shutdown. The following procedures may be used. Shut Down If you are connected to the spectrometer, select Unlink from the Spectrometer Control tool. This will stop communication between the workstation and acquisition system. Next, turn off the power to the console using the main power switch located on the front of the console. Shut down the workstation. Note that it is not usually necessary to shut down the workstation for a system reboot. However, it should be shut down if you expect a power outage. Turn off the monitor and printer. The system is now completely shut down. Start-up Turn on the workstation, monitor and printer. Login to an account set up to run Delta. Launch Delta. Turn on the power to the console using the main power switch. Return to the workstation and monitor the comments in the Delta console. When everything has 71

powered up correctly, you should see "Instrument Ready" in the Master Console. You can now connect to a spectrometer by clicking on the magnet icon. Restarting Control Only If a problem arises in the operation of the spectrometer, it is often not necessary to do a complete system shutdown and restart to remedy the problem. Rather, it is usually only necessary to restart the program that controls the spectrometer. The name of this program is control and it runs on the acquisition computer. Click to disconnect from the spectrometer. On a Windows system, open a command prompt. Then type the following&: telnet eclipse2 root supply the password cd /eclipse/./stop <ENTER> <ENTER> <ENTER> <ENTER> <ENTER> wait a few seconds./start <ENTER> You will notice that the spectrometer will go through its boot sequence. On a Mac Or Linux workstation, open a UNIX shell and type the following: eclipse1$ rlogin -l root eclipse2 supply the password cd /eclipse/./stop <ENTER> <ENTER> <ENTER> <ENTER> wait a few seconds./start <ENTER> You will notice that the spectrometer will go through its boot sequence. ECLIPSE+ NMR Training Guide, v4.3.4 72

VII. NMR System Backup/Restore with Delta Spectrometer Backup There are a number of spectrometer specific files that reside on the acquisition computer. To back these files up, it is possible to do the following: Windows/Mac Workstations running either WINDOWS XP or OSX use a graphical backup tool. On windows systems, this tool resides in: C:\Program Files\JEOL\Delta\bin\Delta Spectrometer Backup.exe To run the backup tool (Figure 71), double-click and follow the prompts. Figure 71 The graphical back/restore tool on Windows On a Mac, this tool resides in Applications -> Delta Utilities. Double-click follow the prompts. and then Linux On Linux based workstations, you need to run the backup tool from the command line. To do this, type the following in a UNIX shell: Note: this assumes the name of your spectrometer is eclipse2./backup_spectrometer eclipse2 <ENTER> Give the root password for the acquisition computer <ENTER> This will create a directory called back_eclispe2 containing the spectrometer files. Alternaytively, one can use the cd_backup script provided on the workstation. To run this script, you need to first log into the root account. Open a UNIX shell. At the prompt, type:./cd_backup ENTER ECA/ECX NMR Training Guide, v4.3-a 73

Follow the instructions as prompted. If you would like to backup the workstation only, be sure and type n when asked if you want to copy spectrometer files. Upon completion of the backup, remove the CD, label it and store it in a safe place. Note that this procedure does not backup any NMR data! VIII. Cryogen Filling The most important maintenance procedure for a superconducting magnet system is to keep the cryostat adequately filled with cryogens. This section gives an OVERVIEW of how to fill both liquid nitrogen and liquid helium on JEOL NMR systems. This section assumes you are somewhat familiar with cryogen fills and that you know how to safely handle cryogenic liquids and compressed gases. If there are any doubts about how to perform these procedures, contact JEOL for advice before attempting a fill! For filling schedules and cryogen volumes for your particular magnet, consult the white Oxford# binder that came with your instrument. Cryogen Sensors Most ECX/ECA NMR systems are equipped with both liquid nitrogen and liquid helium sensors. It is important to remember that these sensors can at times give erroneous results. This is because if moisture or air was introduced into the system during a previous fill, an ice block can form on the sensor. It is therefore recommended to fill cryogens by the calendar and not solely by the reading from a sensor. It is helpful to keep a logbook of cryogen fills, especially helium fills, to ensure that you know when it is time to fill. Additionally, a log file of cryogen sensor readings is stored on the acquisition computer in /usr/eclipse/cryogen.log. This file can be viewed graphically, and plotted, by selecting Tools-> View Log Files -> View Cryogen Log from the Spectrometer Control tool. Nitrogen fills Be sure to order a low-pressure nitrogen dewar (! 22 p.s.i.) for fills. Using a high-pressure transfer dewar can cause irreparable damage to the cryostat! Do not leave the magnet unattended when performing a nitrogen fill! Performing a nitrogen fill is a relatively straightforward task and should be done once a week, every week. It is recommended to choose a day of the week that is not subject to holidays (i.e. not a Monday or a Friday). A nitrogen transfer line can be purchased or constructed using rubber or silicone tubing. Under no circumstances should polyethylene/terphthalate tubing be used. Significant injury can result if this type of tubing breaks at liquid nitrogen temperature. When starting the fill, start slowly until you are sure you are transferring liquid. The fill rate may then be sped up. The fill is completed when liquid nitrogen spews out of the exit port. If the nitrogen continues to surge out of the exit hose after the transfer is stopped, the dewar has been slightly pressurized. This pressurization can be caused by either filling to fast or because their is a ECLIPSE+ NMR Training Guide, v4.3.4 74

blockage in the nitrogen inlet or outlet. For advice concerning this, please contact JEOL Applications or Service. Finally, care should be taken not to spill nitrogen onto the base of the cryostat. Prolonged exposure to cryogens at the base of the magnet can cause a vacuum seal failure. Helium fills Note: It is recommended to fill nitrogen before you do a helium fill. This minimizes atmospheric backflow into the nitrogen during the helium fill. Do not leave the magnet unattended when performing a helium fill! Helium fills are much more demanding than nitrogen fills and should be performed only by experienced personnel. However, the basic concept behind a helium fill is simple. You want to introduce liquid helium only into the cryostat. Liquid helium is pushed out of the helium transfer dewar by a tank of gaseous helium. The gaseous helium should be very pure - no less than 99.997% or U.S. Bureau of Mines Grade A. Remember that compressed gas cylinders are usually magnetic! Keep the tank of helium gas well outside the 5 Gauss line to avoid interaction with the magnet. To help keep room air and moisture from entering the magnet, it is advised to slightly pressurize the cryostat dewar before performing a fill. To do this, you need to first get into console mode. Next, open a new Sample Tool, and under Options choose Helium Fill Charge. This will increase the polling of the helium sensor to every 4 seconds, hence, the result is more boil-off of helium and a positive pressure in the dewar. Do this at least five minutes before performing the fill. Before attempting to insert the transfer line into the magnet, make sure that it has been pre-cooled and is spewing out liquid. Also, make sure that the insulated portion of the transfer line does not get appreciably cold or frosty. If it does, the transfer line may have to be pumped down. Before inserting the transfer line into the cryostat, you will also need to wipe off any snow that has collected on it and stop the flow of helium. Once you have inserted the transfer line and have re-started the flow, you should notice the helium level displayed in charge mode increase to a high level, usually >90%. This indicates that liquid helium is splashing on the sensor. The helium transfer is done when the "plume" coming out of the exit port turns into a "flame". Make sure to exit out of helium charge mode after the fill by closing the Sample Tool. Confirmation that you have exited charge mode will be reported in the Delta Console. Finally, it is also a good idea to recheck the tightness of the bung and exhaust valve after they have reequilibrated to ambient temperature. To check how much liquid is available in a transfer dewar (before or after a fill), it can be "thumped". This procedure uses the "thumper" tube that has a thin rubber membrane on one end. You can make a membrane by fastening a portion of a latex glove on the end with a rubber band. Starting from the bottom of the dewar, a mark can be made on the thumper with a wire tie. Then, if the thumper is raised slowly, you will eventually notice the frequency of vibration felt at the rubber membrane will change. Make a mark at this point with another wire tie. If you measure the distance between the wire ties, you can determine the volume of cryogen in the transfer dewar. See Appendix C or the table provided with your dewar to convert the distance into liters of liquid helium. ECA/ECX NMR Training Guide, v4.3-a 75

Appendix A Shimming the Non-Spin Shims The following procedure outlines a method for shimming the non-spin shims. The shims listed here may not correspond exactly to your shim set. It should be noted that many variations on this procedure are possible. 1) Shim Z1-Z2. 2) Turn off the spinner. 3) Shim X and Y 4) Shim XZ, YZ. If an improvement is made, re-shim X and Y. 5) Shim X2, Y2. If an improvement is made, re-shim X and Y. 6) Shim XZ2, YZ2. If an improvement is made, re-shim X and Y. 7) Shim X2Z, Y2Z. If an improvement is made, re-shim X and Y. 8) Turn the spin on. Shim Z1, Z2. 9) Go to step 2. Repeat this procedure until no further improvement is observed. ECLIPSE+ NMR Training Guide, v4.3.4 76

Appendix B Preparing the System for Gradient Shimming Deuterium Grad Shim Pulsewidth 90 Purpose: Determine the deuterium gradient shim 90 pulse width Sample needed: 5% CHCl 3 with Cr(acac) 3 in acetone d-6 Instrument preparation: Shim the probe to the sample Experiment used: /usr/delta/global/experiments/gradient_shim/single_pulse_2h.exp Process list used: /usr/delta/global/process_lists/std_carbon.list Note: This pulse width is normally measured only once. The initial value can be used for the life of the instrument. On the Header tab, click auto_gain and then Done. Uncheck auto_gain. Next to where it says process, click Edit, Process_Ndimensional and then Accept. Uncheck automatic. Set recvr_gain to 20. Set x_angle to 90[deg]. Make sure that x_atn is set to at least 10[dB]. Submit the experiment. Process the data and copy the offset of the deuterium peak into x_offset. Array x_90_width from 50[us] to 0.8[ms] in steps of 50[us]. Set relaxation_delay to 15[s]. Submit the experiment. Process the result and find your best null. Divide the null by two to get the 2 H obs 90. Figure 72 shows a typical result. Figure 73 highlights where to save your pulse width and attenuator values in the Probe Tool. ECA/ECX NMR Training Guide, v4.3-a 77

Figure 72 2 H Gradient Shim Pulse Width Figure 73 Change the highlighted values to save the 2 H Gradient Shim Pulse Width ECLIPSE+ NMR Training Guide, v4.3.4 78

Generating 2 H Gradient Shimming Basis Sets Purpose: Collect gradient basis sets (calibration maps) Sample needed: 1% CHCl 3 in acetone d-6 lineshape sample Instrument preparation: Roughly shim the probe to the sample Purpose: Collect gradient basis sets (calibration maps) Sample needed: 1% CHCl 3 in acetone d-6 lineshape sample Instrument preparation: Roughly shim the probe to the sample Load the lineshape sample and do a cursory shim, including the non-spin shims. The shimming does not have to be perfect but one should be sure to shim at least Z1, Z2 and the non-spin shims. Open Spectrometer Control -> Config -> Gradient Shim Tool. Click Calibrate. Click Homospoil and 2H (Deuterium). PFG Single should be used for fields at 500Mhz and greater. Click on Z1-Z6 next to where it says Excursion. Click NO DISPLAY so that it says DISPLAY. Click Calibrate. The acquisition of the maps will take some time. If you need to stop the acquisitions, click on Abort! (Figure 74). When Figure 74 Click Abort! to terminate gradient shimming events all of the acquisitions complete, basis sets will appear on the screen. Figure 75 shows a typical result. Change the units on the maps to be in [%]. One can scroll through the units by holding down Shift and clicking i on the keyboard. Zoom the Z3 map to display only the smooth portion of the curve. Zoom the other maps to the same limits. Verify that this % width leaves no jagged or discontinuous regions in the other maps. If it does, readjust the width. Read off the % width covered and enter this value into the Gradient ShimTool where it says % Width. Figure 76 shows the result after zooming the maps. Gradient shim and run the lineshape test to make sure you are happy with the maps and the percent width. If the lineshape test is successful, you should save out the percent width and the gradient basis sets for general use. In the Gradient Shim Tool, click Save next to where it says % Width. This will store the % width in the gradient configure file. The gradient configure file and gradient basis sets (z1-z6) should be saved into /usr/delta/global/instrument, so that all users will have access to these files. It should be noted that Delta only parses the highest numerical increment for gradient basis files and that all the files must reflect the current node name. That is, the node names should be changed if the system is installed on your local network. ECA/ECX NMR Training Guide, v4.3-a 79

Figure 75 Gradient Basis Sets Configuring Delta for Gradient Shimming Integration To complete the gradient shimming configuration process there are some software modifications one should make. The first is on the acquisition computer. Do an rlogin or telnet to the acquisition computer and change to the /eclipse subdirectory. Change machine.pre_exp to correspond to the following example: eclipse2# cd eclipse/ eclipse2# vi machine.pre_exp --Default commands executed before a queued experiment --LOCK_STATE = AUTOLOCK & SHIM LOCK_STATE = AUTOLOCK --AUTOSHIM_MODE = Z1 Z2 eclipse2# Essentially, you need to comment out AUTOSHIM_MODE = Z1 Z2 by adding a double dash to the line. Modifying machine.pre_exp stops auto-shimming from occurring in automation. Finally, you will need to enable gradient shimming in Preferences. Select Master Console -> File -> Preferences and then click. Change Gradient Shim Allowed to True. The gradient shim buttons in the Sample Tool should now be activated (i.e. not grayed out). Also, click to set the automation preferences. Change both Gradient Shim and Gradient Shim Allowed to TRUE. ECLIPSE+ NMR Training Guide, v4.3.4 80

Appendix C Helium Dewar Levels* CMSH-30 CMSH-60 CMSH-100 CMSH-250 CMSH-500 Inches Liters Liters Liters Liters Liters 1 0.80 1.00 1.90 4.00 2.00 2 2.80 3.60 3.52 10.00 7.80 3 5.60 7.50 8.46 16.00 17.30 4 8.80 12.10 13.58 22.00 30.40 5 12.10 17.20 18.70 30.00 47.10 6 15.40 22.30 23.82 39.90 65.50 7 18.70 27.50 28.94 49.80 83.80 8 22.00 32.60 34.06 59.70 102.20 9 25.30 37.80 39.18 69.60 120.50 10 28.50 42.90 44.30 79.50 139.00 11 31.30 48.10 49.42 89.40 157.30 12 33.30 53.20 54.54 99.30 175.60 13 34.10 57.80 59.66 109.20 194.00 14 61.70 64.78 119.10 212.30 15 64.30 69.90 129.00 230.70 16 65.30 75.02 138.90 249.10 17 80.14 148.80 267.40 18 85.26 158.70 285.80 19 90.38 168.60 304.10 20 95.50 178.50 322.50 21 100.42 188.40 340.90 22 104.14 198.30 359.20 23 107.68 208.20 377.60 24 218.10 395.90 25 228.00 414.30 26 237.90 432.70 27 245.90 451.00 28 251.90 469.40 29 257.90 487.70 30 261.90 504.40 31 265.90 517.50 32 267.90 527.00 33 532.80 34 534.80 *Note: This table may not be accurate for all dewars ECA/ECX NMR Training Guide, v4.3-a 81

Appendix D Using the Level Tool Two-dimensional NMR data has a third dimension corresponding to the intensity of the peaks. To represent this dimension on a two-dimensional plot contours are drawn similar to a relief map. The Level Tool is a powerful tool for contouring NMR data. It can be invoked by holding down the right mouse button on a data set. The hot key for invoking the Level Tool is the \ key. A diagram of the Level Tool and its features is shown in Figure 76. The left-most buttons represent the 24 positive and negative Bias Curve Upper Bounds Bias Slider Contours Contour Distribution Peak Intensity Upper Bounds Lower Bounds Contour Level Peak Intensity - + Contour Level Buttons Lower Bounds ECLIPSE+ NMR Training Guide, v4.3.4 Figure 76 Understanding the Level Tool contours, which can be drawn to represent the data. If the Level Tool apears blank, or the data does not respond to changes, make sure the data set of interest is selected by clicking on it with the Page Up key. If a tile is colored and depressed, it has been calculated and is being drawn on the screen. Once tiles have been calculated, they may be turned on and off interactively. The contour level distribution can be changed by adjusting the bias slider. If the bias slider is moved all the way up, the contour distribution will be weighted to draw more slices near the bottom of the peaks. Conversely, if the bias slider is moved all the way down, it will draw more slices at the top of the peaks (Figure 77). To see the changes made by moving the sliders, the Apply button must be clicked to recalculate the result. If you wish to define a different bottom or top of the data, it is possible to change the bounds sliders. Adjusting the bounds sliders can be particularly useful whenever there is a large intensity difference among the peaks in a data set. For example, you may wish to create a greater number contours at the base of a group of peaks while largely excluding the top of a very large peak (Figure 78). 82

Figure 77 The Bias slider Figure 78 The Bounds Sliders To further change the look of contours, single-click-hold the right mouse button the Level Tool. The menu in Figure 79 will appear. Choose Switch to Colors. The Level Tool will change to look something like Figure 80. Click on a colored square and either the plus or minus sign for positive and negative contours, respectively. To apply these changes either ECA/ECX NMR Training Guide, v4.3-a 83

click or. To return to the contour portion of the Level Tool, clickhold the right mouse button and choose Switch to Levels. Figure 79 The Level Tool menu available from the right mouse button Figure 80 The Switch to Colors mode of the Level Tool ECLIPSE+ NMR Training Guide, v4.3.4 84

Appendix E Maintenance Schedule The following maintenance schedule is given to roughly outline a timetable for performing various spectrometer activities. Depending on your site, some of these items may have to be performed more or less frequently. If there are any questions regarding spectrometer upkeep and maintenance, contact JEOL for advice. Weekly Clean spinner(s) with isopropanol. Fill Nitrogen. Monthly Shim the system to specs and save a new system shim file. Remove crash and core dump files from user accounts. As Needed Calibrate pulse widths. Fill helium. Time interval depends on the type of magnet at your site. Refer to the white Oxford! binder for specifics about your system. Remove the probe to clean. Backup and purge data files. Perform Workstation and Acquisition system backup. ECA/ECX NMR Training Guide, v4.3-a 85

Appendix F Probe Tuning Minimizing the Signal on the Reflectance Bridge A probe tune is executed by sending a low power CW signal to the probe. For a probe to be tuned and matched, the probe should absorb as much of this power as possible, and do so as efficiently as possible. One can quantify the goodness of probe tuning by measuring the amount of power being reflected back from the probe with a reflectance bridge. The M-console has a series of green LED s which displays this measurement. Low Power CW Forward Reflected Figure 81 Tuning a probe and the Reflectance Display When the probe tune experiment is running, the Check LED will be illuminated on the M-console. If tuning manually, one would then adjust the tune and match knobs to minimize the signal on the reflectance bridge display. The minimum you observe should be fairly sharp and obvious. Using the Force Tune Flag By default, if the spectrometer senses that the probe is already tuned to a given nucleus, it will not try to tune it again if asked to tune to that nucleus. For example, if the last experiment run on the high-frequency coil was proton, and you ask it to tune to proton, it will indicate that it is not necessary to tune the probe. The Force Tune flag is used to tell the spectrometer to continue with a probe tune, regardless of its last tuned state. Click the box, next to where is says Force Tune, before executing an experiment. When the experiment is submitted, auto-tune probes will start the tuning process. If you are using a manually tuned probe, you will be prompted to tune. This prompt will include the starting tune and match numbers and appropriate capacitor stick (if needed). Figure 81 shows an example of what you would see when tuning to phosphorus. Click finished tuning. when you are ECLIPSE+ NMR Training Guide, v4.3.4 86

Figure 81 The probe tuning dialog box ECA/ECX NMR Training Guide, v4.3-a 87